Endothelin DNA and use thereof

ABSTRACT

Disclosed are (1) a DNA sequence containing a segment coding for endothelin-2 or human endothelin-3, (2) a precursor protein and a mature peptide of endothelin-2 or human endothelin-3, (3) a transformant carrying a DNA sequence containing a DNA segment coding for endothelin-2 or human endothelin-3 and (4) a method for preparing mature endothelin-2 or endothelin-3 which comprises culturing the transformant described in (3), producing and accumulating a protein in a culture medium, and collecting the same. 
     Cells transfected or transformed with the DNA allow large amounts of endothelin-2 or endothelin-3 to be produced, which causes the advantageous production of endothelin-2 or endothelin-3. 
     Endothelin-2 and endothelin-3 can be utilized as hypotension therapeutic agents or local vasoconstrictors to animals including humans.

This is a continuation of copending application(s) Ser. No. 07/422,132filed on Oct. 16, 1989, now U.S. Pat. No. 5,231,166.

BACKGROUND OF THE INVENTION

The present invention relates to a DNA sequence containing a DNA segmentcoding for a human vasoconstrictive peptide, namely endothelin-2, aprecursor protein (or a precursor polypeptide) and a mature protein (ora mature polypeptide) of endothelin-2 and a method for preparing theprecursor protein and the mature protein (endothelin-2), and further toa DNA sequence containing a DNA segment coding for human endothelin-3, aprecursor protein of human endothelin-3 and a method for preparingendothelin-3.

In this specification, the term "precursor protein" is preferably usedto describe a protein which includes an amino acid sequence of a maturepeptide and has a portion or all of an amino acid sequence coded with aDNA segment of the peptide at the N-terminus, the C-terminus or bothtermini thereof.

There have been reports of endothelium-dependent vasoconstrictorreactions to various mechanical and chemical stimuli as well asendothelium-dependent vasodilative reactions. For example, it is knownthat vasoconstriction can be induced by mechanical loads such asvascular stretch and increased vascular inner pressure, or can bechemically induced by such agents as thrombin. Further, vasoconstrictioncan be induced by conditions of anoxia. Noradrenaline-inducedvasoconstriction can be enhanced by use of neuropeptide Y [K. Takemoto,Proc. Natl. Acad. Sci. U.S.A. 79, 5485 (1982); C. Minth et al., ibid.81, 4577 (1984)]. Endothelial cell-derived coronary vascular constrictorfactors (each having molecular weights of 8,500 and 3,000) are describedin K. A. Hickey et al., Am. J. Physiol. 248, C550(1985); and in R. F.O'Brien, J. Cell Physiol. 132, 263 (1987). However, their structures areunknown. An endothelial cell-derived peptide-like substance is alsodescribed in M. N. Gillespie et al., J. Pharmac. Exp. Ther. 236, 339(1985). However, the structure of that substance is also unknown.

Vasopressin is known as a peptide having a vasoconstrictor activity, andthe amino acid sequence thereof was determined. There have been noreports, however, that vasopressin was obtained from mammalian or birdvascular endothelial cells. Although there is a report that anangiotensin having a vasoconstrictor activity was obtained from theendothelial cells of bovine aortas [I. Kifor and V. J. Dzav, Circ. Res.60, 422 (1987)], the angiotensin is a peptide having a molecular weightof only about 1,000.

Some of the present inventors have previously succeeded in isolatingporcine endothelin as a peptide having a similar vasoconstrictoractivity from the endothelial cells of porcine aortas (JapaneseUnexamined Patent Publication No. 206997/1989). Some of the presentinventors have also succeeded in isolating human endothelin and cloningporcine endothelin cDNA and human endothelin cDNA (Japanese patentapplication Nos. 275613/1987, 313155/1987, 148158/1988 and 274454/1988).The mature polypeptides of the porcine endothelin and the humanendothelin have the same amino acid sequence, and are referred to asendothelin-1.

Further, the present inventors have filed patent applications withrespect to the isolation of rat endothelin and the cloning of its cDNA(Japanese patent application Nos. 174935/1988 and 188083/1988), and thisrat endothelin is referred to as endothelin-3.

Furthermore, the present inventors have also filed a patent applicationwith respect to the isolation of mouse endothelin and the cloning of itscDNA (Japanese patent application No. 223389/1988), and this mouseendothelin is referred to as endothelin B.

The amino acid sequences of the endothelin-1, endothelin B andendothelin-3 are shown in FIG. 3 in comparison to one another.

Endothelin is a general term for peptides having a molecular weight of2500+300 and having 21 amino acid residues, including four cysteinegroups located at the lst, 3rd, 11th and 15th residues from theN-terminus of the amino acid sequence, which form two sets of disulfidebonds. One of the combinations of the disulfide bonds may be 1-15 and3-11 cysteine groups, and the other may be 1-11 and 3-15. The former ishigher in ratio of formation and in activity than the latter.

These above-described endothelins have been called variously, and in thepresent invention, newly unified names for the endothelins are employedin the present invention. They are shown as compared with the previousnames as follows:

    ______________________________________                                        Newly unified names Previous names                                            ______________________________________                                        endothelin-1        endothelin A                                                                  human endothelin                                                              porcine endothelin                                                            endothelin α                                        endothelin-B        mouse endothelin                                                              endothelin B                                                                  endothelin β                                         endothelin-3        endothelin C                                                                  endothelin γ                                                            rat endothelin                                            ______________________________________                                    

As described above, homologous endothelin peptides have been discoveredfrom various animals. However, no novel hologous genes have beendiscovered from the same animal species. It is therefore a currentsubject that novel homologous endothelin is further screened, and thestructure and activity of the endothelin is studied, thereby examiningits usefulness, and that the novel peptide is cloned by generecombination to pioneer mass production thereof.

SUMMARY OF THE INVENTION

The present inventors have variously studied, considering that importantcontributions will be made to future studies and medical treatments, ifa novel homologous gene having the vasoconstrictor activity describedabove can be collected and further prepared by gene recombination. As aresult, the following information has been obtained, thus arriving atthe present invention.

Namely, the present inventors have succeeded in cloning DNA coding forendothelin having an amino acid sequence different from that of theabove endothelin-1[human endothelin (endothelin A)]from a human genomicDNA library by using as a probe the synthesized DNA segment coding for apart of the human endothelin described in the patent applicationspreviously filed. The present inventors have also succeeded inpioneering the mass production of the new endothelin by generecombination. The present inventors have named this human endothelinhaving the novel amino acid sequence "endothelin-2" (first named "humanendothelin A-II").

In the cloning described above, the present inventors have furtherdiscovered a new human eldothelin DNA which is the same as the aboveendothelin-3[rat endothelin (endothelin C)] in amino acid sequence ofthe mature protein, but different therefrom in nucleotide sequencecoding for the mature protein and in precursor amino acid sequence. Thepresent inventors have named this DNA "human endothelin-3 DNA", and theprecursor "human endothelin-3 precursor protein".

In accordance with the present invention, there are provided (1) a DNAsequence containing a DNA segment coding for endothelin-2, (2) aprecursor protein and a mature peptide of endothelin-2, (3) atransformant carrying a DNA sequence containing a DNA segment coding forendothelin-2 and (4) a method for preparing mature endothelin-2 whichcomprises culturing the transformant described in (3), producing andaccumulating a protein in a culture medium, and collecting the proteinthus obtained. There are further provided (5) a DNA sequence containinga DNA segment coding for human endothelin-3, (6) a human endothelin-3precursor protein, (7) a transformant carrying a DNA sequence containinga DNA segment coding for human endothelin-3 and (8) a method forpreparing mature endothelin-3 which comprises culturing the transformantdescribed in (7), producing and accumulating a protein in a culturemedium, and collecting the protein thus obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows simplified restriction enzyme maps of a DNA sequencecontaining an endothelin-2 precursor or mature peptide DNA segment andof a DNA sequence containing a human endothelin-3 DNA segment;

FIG. 2 shows a nucleotide sequence of the endothelin-2 precursor ormature peptide DNA segment, a nucleotide sequence of the humanendothelin-3 DNA segment, nucleotide sequences of endothelin-1 and ratendothelin-3 as comparative examples, and their amino acid sequencespresumed therefrom; and

FIG. 3 shows an amino acid sequence of the human endothelin-2 maturepeptide presumed from the nucleotide sequence shown in FIG. 2, and aminoacid sequences of endothelin-1, endothelin B and endothelin-3.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Endothelin-2 precursor of the present invention comprises the followingamino acid sequence [formula (2)]. ##STR1##

Mature endothelin-2 of the present invention corresponding tohuman-derived mature endothelin (endothelin-1) and consisting of 21amino acid residues has an amino acid sequence represented by thefollowing formula (2') which corresponds to No. 12 to 32 of the formula(2): ##STR2##

The numbers of the amino acid residues in the above formula are given inorder starting from the first Cys with respect to the amino acidsequence of mature endothelin-2, and different from the numbers given toa endothelin-2 precursor in the formula (2).

Endothelin-2 is different from endothelin-1 in the amino acid residuesunderlined in the above formula and has the amino acid residues of##STR3## in endothelin-1.

A DNA sequence of the present invention coding for endothelin-2 containsa nucleotide sequence represented by the following formula (1) or aportion thereof: ##STR4## This DNA sequence is widely different fromthose of the known endothelin-1, endothelin B and endothelin-3 as shownin FIG. 2.

Further, a DNA sequence of the present invention coding for humanendothelin-3 has a nucleotide sequence represented by the followingformula (3) or a portion thereof: ##STR5## It is as shown in FIG. 2 thatthis DNA sequence is also different from the known sequences and isnovel.

The DNA sequence corresponding to mature proteins [corresponding to Nos.34 to 96 in formulas (1) and (3)] are also different from the knownendothelin DNA sequences, and hence the DNA sequences of the presentinvention are novel.

Human endothelin-3 precursor comprises the following amino acid sequencerepresented by the formula (4), which differs from the amino acidsequence of rat endothelin-3 precursor as shown in FIG. 2 and which isnovel. ##STR6##

As the DNA sequences of the present invention coding for theendothelin-2 mature peptide (endothelin-2), any DNA sequence may be usedas long as a DNA sequence contains a nucleotide sequence coding for theamino acid sequence [Nos. 12 to 32 in formula (2)] of the endothelin-2mature peptide. For example, a DNA sequence containing the nucleotidesequence represented by formula (1) or a portion thereof is preferablyused.

The nucleotide sequence represented by formulat (1) is the endothelin-2DNA sequence obtained in the present invention. The endothelin-2 aminoacid sequence depicted in formula (2') corresponds to the nucleotidesequence represented by Nos. 34 to 96 in formula (1).

In the present invention, for example, an expression vector having theDNA sequence containing the nucleotide sequence coding for matureendothelin-2 can be prepared by the following process:

(a) Messenger RNA (mRNA) is isolated from endothelin-2-producing cells.

(b) Single stranded complementary DNA (cDNA) is synthesized from themRNA, followed by synthesis of double stranded DNA.

(c) The complementary DNA is introduced in a cloning vector such as aphage or a plasmid.

(d) Host cells are transformed with the recombinant phage or plasmidthus obtained.

(e) After cultivation of the transformants thus obtained, plasmids orphages containing the desired DNA are isolated from the transformants byan appropriate method such as hybridization with a DNA probe coding fora portion of endothelin-2 or immunoassay using an anti- endothelin-2antibody.

(f) The desired cloned DNA sequence is cut out from the recombinant DNA.

(g) The cloned DNA sequence or a portion thereof is ligated downstreamfrom a promoter in the expression vector.

The mRNA coding for endothelin-2 can be obtained from variousendothelin-producing cells such as endothelial cells of human aortas andhuman placentas.

Methods for preparing the mRNA from the endothelin-2-producing cellsinclude the guanidine thiocyanate method [J. M. Chirgwin et al.,Biochemistry 18, 5294 (1979)]and the like.

Using the mRNA thus obtained as a template, cDNA is synthesized by useof reverse transcriptase, for example, in accordance with the method ofH. Okayama et al. [Molecular and Cellular Biology 2, 161 (1979); ibid.3, 280 (1983)]. The cDNA thus obtained is introduced into the plasmid.

The plasmids into which the cDNA may be introduced include, for example,pBR322 [Gene 2, 95 (1977)], pBR325 [Gene 4, 121 (1978)], pUC12 [Gene 19,259 (1982)]and pUC13 [Gene 19, 259 (1982)], each derived fromEscherichia coli, and pUB110 derived from Bacillus subtilis [Biochemicaland Biophysical Research Communication 112, 678 (1983)]. However, anyother plasmid can be used as long as it is replicable and growable inthe host cell. Examples of the phage vectors into which the cDNA may beintroduced include λgtll [R. Young and R. Davis, Proc. Natl. Acad. Sci.U.S.A. 80, 1194 (1983)]. However, any other phage vector can be used aslong as it is growable in the host cell.

Methods for introducing the cDNA into the plasmid include, for example,the method described in T. Maniatis et al., Molecular Cloning, ColdSpring Harbor Laboratory, p.239 (1982). Methods for introducing the cDNAin the phage vector include, for example, the method of T. V. Hyunh etal. [DNA Cloning, A Practical Approach 1, 49 (1985)].

The plasmid thus obtained is introduced into an appropriate host cellsuch as Escherichia and Bacillus.

Examples of Escherichia described above include Escherichia coli K12DHl[Proc. Natl. Acad. Sci. U.S.A. 60, 160 (1968)], M103 [Nucleic AcidsResearch 9, 309 (1981)], JA221 [Journal of Molecular Biology 120, 517(1978)]HB101 [Journal of Molecular Biology 41, 459 (1969)]and C600[Genetics 39, 440 (1954)].

Examples of Bacillus described above include Bacillus subtilis MI114[Gene 24, 255 (1983)] and 207-21 [Journal of Biochemistry 95, 87(1984)].

Methods for transforming the host cell with the plasmid include, forexample, the calcium chloride method or the calcium chloride/rubidiumchloride method described in T. Maniatis et al., Molecular Cloning, ColdSpring Harbor Laboratory, p.249 (1982).

When a phage vector is used, for example, the phage vector can betransduced into multiplied Escherichia coli, using the in vitropackaging method.

Human cDNA libraries containing endothelin-2 cDNA can be obtained bynumerous techniques well known in the art including purchasing them fromthe market, though obtainable by the methods described above. Forexample, a cDNA library from human placentas is available from ClontechLaboratories, Inc., U.S.A.

Methods for cloning an endothelin-2 DNA from the human DNA libraryinclude, for example, the plaque hybridization method usingoligonucleotides chemically synthesized on the basis of phage vectorλcharon 4A and the amino acid sequence of endothelin-2 as a probe [T.Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory,(1982)]. The endothelin-2 DNA thus cloned is subcloned in, for example,pBR322, pUC12, pUC13, pUC19, pUC118 and pUC119 to obtain theendothelin-2 DNA, if necessary.

The nucleotide sequence of the DNA sequence thus obtained is determinedby, for example, the Maxam-Gilbert method [A. M. Maxam and W. Gilbert,Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1977)] or the dideoxy method [J.Messing et al., Nucleic Acids Research 9, 309 (1981)], and the existenceof the endothelin-2 DNA is confirmed in comparison with the known aminoacid sequence.

As described above, the DNA sequence [endothelin-2 DNA, represented byformula (1)]coding for endothelin-2 is obtained.

FIG. 1 shows the restriction enzyme fragment maps of the DNA sequencecontaining the DNA segment coding for endothelin-2 obtained in Example 2described below and of the DNA sequence containing the humanendothelin-3 DNA obtained in Example 3 described below. FIG. 2 shows thenucleotide sequences of the DNA sequences as determined by the dideoxymethod, and FIG. 3 shows the amino acid sequences ascertained from thenucleotide sequences.

The DNA sequence coding for endothelin-2 cloned as described above canbe used as it is, or after digestion with a restriction enzyme ifdesired, depending on the intended use.

A region intended to be expressed is cut out from the cloned DNA andligated downstream from the promoter in a vehicle (vector) suitable forexpression, whereby the expression vector can be obtained.

The DNA sequence has ATG as a translation initiating codon at the5'-terminus thereof and may have TAA, TGA or TAG as a translationterminating codon at the 3'-terminus. The translation initiating codonand translation terminating codon may be added by use of an appropriatesynthetic DNA adaptor. A promoter is further ligated in the upstreamthereof for the purpose of expressing the DNA sequence.

Examples of the vectors include the above plasmids derived fromEscherichia coli such as pBR322, pBR325, pUC12, and pUC13, the plasmidsderived from Bacillus subtilis such as pUB110, pTP5 and pC194, plasmidsderived from yeast such as pSH19 and pSH15, bacteriophage such asλphage, and animal viruses such as retroviruses and vaccinia viruses.

As the promoter used in the present invention, any promoter isappropriate as long as the promoter is suitable for expression in thehost cell selected for the gene expression.

When the host cell used for transformation is Escherichia, it ispreferable that a trp promoter, a lac promoter, a recA promoter, a λPLpromoter, a lpp promoter, etc. are used. When the host cell is Bacillus,it is preferable that a PH05 promoter, a PGK promoter, a GAP promoter,an ADH promoter, etc. are used. In particular, it is preferable that thehost cell is Escherichia and the promoter is the trp promoter or the λPLpromoter.

When the host cell is an animal cell, a SV-40 derived promoter, aretrovirus promoter, a metallothionein promoter, a heat shock promoter,etc. are each usable.

An enhancer, a certain DNA sequence important for promoter's activity ina cell, is also effectively used for expression.

Using a vector containing the DNA sequence coding for the endothelin-2mature peptide (endothelin-2) thus constructed, transformants areprepared.

The host cells include, for example, Escherichia, Bacillus, yeast andanimal cells.

As examples of the above Escherichia and Bacillus, strains similar tothose described above can be mentioned.

Examples of the above yeast include Saccharomyces cerevisiae AH22,AH22R⁻, NA87-11A and DKD-5D.

Examples of the animal cells include monkey cell COS-7, Vero, Chinesehamster cell (CHO), mouse L cell and human FL cell.

The transformation of the above Escherichia is carried out according to,for example, the method described in Proc. Natl. Acad. Sci. U.S.A. 69,2110 (1972) or Gene 17, 107 (1982).

The transformation of the above Bacillus is conducted according to, forexample, the method described in Molecular & General Genetics 168, 111(1979).

The transformation of the yeast is carried out according to, forexample, the method described in Proc. Natl. Acad. Sci. U.S.A. 75, 1929(1978).

The transformation of the animal cells is carried out according to, forexample, the method described in Virology 52, 456 (1973).

Thus, there are obtained transformants transformed with an expressionvector containing the DNA sequence coding for the endothelin-2 maturepeptide (endothelin-2).

When bacterial transformants are cultured, a liquid medium isparticularly suitable as a medium used for culture. Carbon sources,nitrogen sources, inorganic compounds and others necessary for growth ofthe transformant are contained therein. Examples of the carbon sourcesinclude glucose, dextrin, soluble starch and sucrose. Examples of thenitrogen sources include inorganic or organic materials such as ammoniumsalts, nitrates, corn steep liquor, peptone, casein, meat extracts,soybean meal and potato extract solution. The inorganic compoundsinclude, for example, calcium chloride, sodium dihydrogenphosphate andmagnesium chloride. Yeast extract, vitamins, growth promoting factorsand so on may be further added thereto.

The pH of the medium is preferably about 5 to 8.

As the medium used for cultivation of Escherichia, there is preferred,for example, M9 medium containing glucose and Casamino Acids (Miller,Journal of Experiments in Molecular Genetics 431-433, Cold Spring HarborLaboratory, New York, 1972). In order to make the promoter actefficiently, a drug such as 3-indolylacrylic acid may be added theretoif necessary.

When the host cell is Escherichia, the cultivation is usually carriedout at about 15° to 43° C. for about 3 to 24 hours, with aeration oragitation if necessary.

When yeast transformants are cultured, there is used, for example,Burkholder minimum medium [K. L. Bostian et al., Proc. Natl. Acad. Sci.U.S.A. 77, 4505 (1980)] as the medium. The pH of the medium ispreferably adjusted to about 5 to 8. The cultivation is usually carriedout at about 20° to 35° C. for about 24 to 72 hours, with aeration oragitation if necessary.

When animal cell transformants are cultured, examples of the mediumsinclude MEM medium containing about 5 to 20% fetal calf serum [Science122, 501 (1952)], DMEM medium [Virology 8, 396 (1959)], RPMI1640 medium(Journal of the American Medical Association 199, 519 (1967)]and 199medium [Proceeding of the Society for the Biological Medicine 73, 1(1950). The pH is preferably about 6 to 8. The cultivation is usuallycarried out at about 30° to 40° C. for about 15 to 60 hours, withaeration or agitation if necessary.

The endothelin-2 mature peptide (endothelin-2) can be isolated andpurified from the culture described above, for example, by the followingmethod.

When the endothelin-2 mature peptide is to be extracted from thecultured cells, the cells are collected by methods known in the artafter cultivation. Then, the collected cells are suspended in anappropriate buffer solution and disrupted by ultrasonic treatment,lysozyme and/or freeze-thawing. Thereafter, a crude extracted solutionof the endothelin-2 mature peptide is obtained by centrifugation orfiltration. The buffer solution may contain a protein denaturant such asurea or guanidine hydrochloride, or a surface-active agent such asTriton X-100.

When the endothelin-2 precursor protein or mature peptide is secreted inthe culture solution, a supernatant is separated from the cells bymethods known in the art after the conclusion of cultivation, and thencollected.

The separation and purification of the endothelin-2 precursor protein ormature peptide contained in the culture supernatant or the extractedsolution thus obtained can be performed by an appropriate combination ofknown separating and purifying methods. The known separating andpurifying methods include methods utilizing solubility such as saltprecipitation and solvent precipitation, methods mainly utilizing adifference in molecular weight such as dialysis, ultrafiltration, gelfiltration and SDS-polyacrylamide gel electrophoresis, methods utilizinga difference in electric charge such as ion-exchange columnchromatography, methods utilizing specific affinity such as affinitychromatography, methods utilizing a difference in hydrophobicity such asreverse phase high performance liquid chromatography and methodsutilizing a difference in isoelectric point such as isoelectro-focussingelectrophoresis.

The activity of the endothelin-2 precursor protein or mature peptidethus formed can be measured by an enzyme immunoassay using a specificantibody. If the products have vasoconstrictive activity, this activitymay also be measured as an index.

The methods for cloning endothelin-2 DNA and producing endothelin-2protein, hereinbefore described, are similarly applicable for cloning ofhuman endothelin-3 DNA and production of human endothelin-3 protein.

The cells, such as animal cells or Escherichia coli, transfected ortransformed with the DNA sequence of the present invention allow largeamounts of the endothelin-2 or endothelin-3 mature peptide to beproduced. Hence, the production of these peptides can be advantageouslyachieved.

The endothelin-2 and endothelin-3 mature peptides prepared here not onlycan be utilized as hypotension therapeutic agents or localvasoconstrictors, but also give a clue to analysis of the mechanism ofthe vasoconstrictor reactions in vivo and to elucidation of antagoniststo the vasoconstrictor factors, including the other endothelin peptides.Endothelin-2 has vasoconstrictor activity up to twice as high as thepreviously isolated endothelin peptides. These peptides have sucheffects as preventing various kinds of hemorrhage, for example, gastricor esophageal hemorrhage as vasoconstrictors, and may also be useful incuring various shock symptoms. The peptides can be administered orally,locally, intravenously or parenterally, preferably locally orintravenously. The dose is 0.001 μg to 100 μg/kg, preferably 0.01 μg to10 μg/kg. The dose is preferably dependent on weight and preferably usedin the form of a solution in 1 to 10 ml of a saline solution.

The peptides of the present invention can be formed into variouspreparations together with additional components, such as emulsions,hydrated mixtures, tablets, solutions, powders, granules, capsules andpills. Examples of the additional components include pharmaceuticallyacceptable vehicles, disintegrators, lubricants, binders, dispersants,plasticizers, fillers and carriers. As to the additional components,examples of the vehicles include lactose, glucose and white sugar; thoseof the disintegrators include starch, sodium alginate, agar powder andcarboxymethyl cellulose calcium; those of the lubricants includemagnesium stearate, talc and liquid paraffin; those of the bindersinclude syrup, gelatin solution, ethanol and polyvinyl alcohol; those ofthe dispersants include methyl cellulose, ethyl cellulose and shellac;and those of the plasticizers include glycerin and starch. Whennucleotides, amino acids and so on are indicated by the abbreviations inthis specification and drawings, the abbreviations adopted by IUPAC-IUBCommission on Biochemical Nomenclature or commonly used in the art areemployed. For example, the following abbreviations are used. When aminoacids are capable of existing as optical isomers, the L-forms arerepresented unless otherwise specified.

DNA : Deoxyribonucleic acid

cDNA : Complementary deoxyribonucleic acid

A : Adenine

T : Thymine

G : Guanine

C : Cytosine

RNA : Ribonucleic acid

mRNA : Messenger ribonucleic acid

dATP : Deoxyadenosine triphosphate

dTTP : Deoxythymidine triphosphate

dGTP : Deoxyguanosine triphosphate

dCTP : Deoxycytidine triphosphate

ATP : Adenosine triphosphate

EDTA : Ethylenediaminetetraacetic acid

SDS : Sodium dodecyl sulfate

Gly or G : Glycine

Ala or A : Alanine

Val or V : Valine

Leu or L : Leucine

Ile or I : Isoleucine

Ser or S : Serine

Thr or T : Threonine

Cys of C : Cysteine

Met or M : Methionine

Glu or E : Glutamic acid

Asp or D : Aspartic acid

Lys or K : Lysine

Arg or R : Arginine

His or H : Histidine

Phe or F : Phenylalanine

Tyr or Y : Tyrosine

Trp or W : Tryptophan

Pro of P : Proline

Asn or N : Asparagine

Gln or Q : Glutamine

With respect to the endothelin-2 mature peptide and human endothelin-3of the present invention, a portion of the amino acid sequence may bemodified, namely there may be addition, elimination or substitution withother amino acids as long as the vasoconstrictor property is not lost.

The present invention will hereinafter be described in detail with thefollowing Reference Example and Examples. It is understood that theReference Example and Examples are not intended to limit the scope ofthe invention. Transformant Escherichia coli XL-1/pghET20SG1 obtained inExample 2 and transformant Escherichia coli XL-1/pghET3E1 obtained inExample 3 are deposited in Fermentation Research Institute, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, Japan (FRI) with the accession numbers FERM BP-2118 and FERMBP-2119, respectively, on October 24, 1988.

Reference Example

(1) Assay of Vascular Smooth Muscle Constrictor Activity

Porcine right coronary artery spiral specimens (0.5×20 mm) with theintima denuded by rubbing with a small swab are suspended in 3 ml ofKrebs-Ringer solution maintained at 37° C. and saturated with a mixedgas containing 5% carbon dioxide and 95% oxygen by volume. After settingthe basal tension to 2 g, the isometric tension is measured with tensiontransducers.

(2) Assay of Cardiotonic Action

Instead of the porcine right coronary artery spiral specimens used inthe assay described in the above item (1), suspended guinea pig rightatrium specimens are used, and the tension and the heart rate per minuteare measured according to the same procedure as described in (1).

Example 1

Preparation of DNA Probe Coding for a Portion of Endothelin-1 [PorcineEndothelin (Human Endothelin I)]

The messenger RNA sequence expected from the amino acid sequencecomposed of the 7th to the 16th residues of endothelin-1,

Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile, was used tochemically synthesize a DNA probe having the following sequence.

^(5') ATG GAC AAG GAG TGT GTC TAC TTC TGC CAT CTG GAC ATC ATC^(3')

The 5'-terminus of this DNA probe was phosphorylated with [μ-³² p]ATP byusing T4 polynucleotide kinase. The phosphorylated DNA probe was usedfor screening a genomic DNA library.

Example 2

Isolation of Endothelin-2 Precursor Genomic DNA and Determination ofNucleotide Sequence Thereof

Escherichia coli Le392 was infected with the above human genomic DNAlibrary (Clontech Laboratories, Inc.) and plated, whereby phage plaqueswere allowed to appear. According to the report of W. Benton and R.Davis [Science 196, 180-182 (1977)], a portion of plaque DNA wastransferred to a nylon filter and hybridized with the DNA probe labeledwith ³² P in Example 1. The hybridization was carried out in thepresence of 20% formamide at 42° C., and then the filter was washed in0.2 X SSC, 0.1% SDS at 20° C. Hybridization-positive clones wereisolated. Then, a mature code region of μghET20, one of these clones,was cut out with SacI and subcloned in plasmid pUC118. By transformingEscherichia coli XL-1 with the resulting plasmid, transformantEscherichia coli XL-1/pghET20SG1 was obtained. There is shown in FIG. 1the simplified restriction enzyme map of a human genomic DNA fragmentcontained in this plasmid. In the FIGURE, the heavy bar ( ) shows amature endothelin-2 code region.

This mature peptide code region and the nucleotide sequence in thevicinity thereof were determined by the method of Sanger [Proc. Nat.Acad. Sci. U.S.A. 74, 5463-5467 (1977)]. The nucleotide sequence and theamino acid sequence presumed therefrom are shown in FIG. 2. The regionsurrounded by shows the mature peptide portion. FIG. 3 shows, forcomparison, the amino acid sequences of the mature peptides ofendothelin-1, endothelin B and rat endothelin-3 which have previouslybeen discovered, along with the amino acid sequence of the endothelin-2mature peptide.

Example 3

Isolation of Mature Human Endothelin-3 Code Region and Determination ofNucleotide Sequence Thereof

Using methods similar to those in Example 2, hybridization-positiveclones were isolated. Then, a mature code region of λghET3, one of theseclones, was cut out with EcoRI and subcloned in plasmid pUC118. Bytransforming Escherichia coli XL-1 with the resulting plasmid,transformant Escherichia coli XL-1 with the resulting plasmid,transformant Escherichia coli XL-1/pghET3E1 was obtained. There is shownin FIG. 1 the simplified restriction enzyme map of a human genomic DNAfragment contained in this plasmid. In the FIGURE, the heavy bar ( )shows a mature human endothelin-3 code region.

This mature peptide code region and the nucleotide sequence in thevicinity thereof were determined by the method of Sanger [Proc. Nat.Acad. Sci. U.S.A. 74, 5463-5467 (1977)]. The nucleotide sequence and theamino acid sequence presumed therefrom are shown in FIG. 2. The regionsurrounded by a frame shows the endothelin-3 mature peptide portion.

Example 4

(1) Synthesis of Endothelin-2

Human endothelin A-II was synthesized by a conventional method, using0.7 g (0.5 m mol) of the commercially available Boc-Trp(CHO)-PAM resin(Applied Biosystems) and a peptide synthesizer (Applied Biosystems,Model 430A).

A condensation process was conducted as follows:

A Boc group on the resin was treated with 50% trifluoroacetic acid inmethylene chloride to form a free terminal amino group, to which thefollowing amino acids were condensed in turn according to the amino acidsequence of endothelin-2 from the C-terminus in the presence ofdicyclohexyl carbodiimide (DCC);

Boc-Ile, Boc-Asp(OBzl), Boc-Leu, Boc-His(Tos), Boc-Cys(Acm),Boc-Tyr(Br-z), Boc-Val, Boc-Phe, Boc-Glu(OBzl), Boc-Lys(Cl-Z),Boc-Trp(CHO) and Boc-Ser(Bzl)

890 mg of 2.53 g of the protected endothelin-2 resin thus obtained wasswelled with 1 ml of anisole and 1 ml of 1,2ethanedithiole, and thentreated with 10 ml of hydrogen fluoride at 0° C. for 60 minutes,followed by removal of excess hydrogen fluoride under reduced pressure.After washing with 5 ml of diethyl ether, the residue was extracted withtrifluoroacetic acid and the resin was filtered off. After removal oftrifluoroacetic acid by distillation under reduced pressure, the extractwas dissolved in 50% aqueous acetic acid and applied on a dextran gelcolumn (Sephadex G-50, 2×90 cm). Peak fractions eluted with the abovesolvent were collected and lyophilized, thereby obtaining 180 mg ofwhite powder. In 4 ml of 50% aqueous acetic acid was dissolved 31 mg ofthe powder, and 19 mg of trifluoroacetate-mercury(II) was added thereto,followed by stirring at room temperature for 16 hours. The resultingmixture was diluted by addition of 100 ml of n-butanol, 50 ml ofmethanol and 50 ml of water, and hydrogen sulfide gas was blown throughit. Then, 5% NH₀ H was added thereto for adjustment to pH 8, andthereafter the mixture was subjected to air-oxidation for 6 hours.Acetic acid was further added thereto to give pH 3, followed bylyophilization. The lyophilized mixture was applied to a Sephadex G-50column (2×90 cm) filled with 50% acetic acid, and the peak fractionswere collected. The collected fractions were further fractionated byusing HPLC [column: YMC (YMC Co. Ltd.), solvent: eluted by a lineargradient of 0.1% aqueous trifluoroacetic acid and acetonitrilecontaining 0.1% trifluoroacetic acid]to obtain 1.0 mg of the desiredproduct.

Synthesized endothelin-2 was eluted by HPLC at 41.4 minutes, in contrastwith 40.2 minutes for endothelin-1.

Column conditions

Wakosil 5C18 (Wako Chem. Ind. Ltd., Japan) (4.6×250 mm)

Eluent; A (0.1% aqueous trifluoroacetic acid) B (acetonitrile containing0.1% trifluorioacetic acid)

Linear concentration gradient elution from A to B (50 minutes)

Flow rate: 1.0 ml/minutes Values of analysis of amino acids: Values ofanalysis (the number in the synthesized product)

    ______________________________________                                        Asx 1.92 (2)  Ser 2.31 (3) Glx 1.04 (1)                                       Cys 1.30 (1)  Val 1.16 (1) Ile 1.35 (1)                                       Leu 1.85 (2)  Tyr 0.89 (1) Phe 1.01 (1)                                       His 1.10 (1)  Lys 1.03 (1) Trp (*) (2)                                        ______________________________________                                         * No data was obtained due to acid decomposition.                        

The combination of the disulfide bonds was 1-15 and 3-11 cysteine groupsin mature endothelin-2.

(2) Assay

The activity of endothelin-2 obtained in the above item (1) was assayedby the method of the Reference Example.

ED₅₀ (median effective dose: effective dose for 50% of tested animals)measured by the assay (1) using porcine coronary artery was 8×10⁻¹⁰ to10×10⁻¹⁰ mol/1.

(3) Injection preparation

12 μg of endothelin-2 obtained in (1) was dissolved in a saline solutionand then filtered by a Millipore filter, followed by lyophilization. Anintravenous injection preparation was prepared by dissolving thelyophilized produced in a saline solution to a total volume of 5 ml foruse.

The following references, which are referred to for their disclosures atvarious points in this application, are incorporated herein byreference.

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What is claimed is:
 1. A DNA molecule comprising a DNA sequence whichencodes a endothelin-2 protein having the amino acid sequence: ##STR7##2. The DNA molecule of claim 1, wherein said DNA sequence has thenucleotide sequence: ##STR8##
 3. A host cell which is transformed by theDNA molecule of claim 1 or
 2. 4. A method of producing matureendothelin-2 protein which comprises culturing the host cell of claim 3,allowing mature endothelin-2 to accumulate in the culture medium, andpurifying the mature endothelin-2 protein.